The present invention is related to a novel process for accumulating and producing desired product molecules. More particularly, the present invention includes a process for accumulating chimeric molecules capable of forming oligomers in or within a portion of a lipid bilayer or the cytoplasm.
General limitations on commercial production of compounds include the efficiency of synthesis and, if a complex synthesis process is involved, the efficiency of recovery. Polypeptide production is an example of the limitations that can effect commercial production. The advent of recombinant DNA techniques has enabled investigators to produce substantial amounts of desired polypeptides in a variety of expression systems (e.g., bacterial, fungal, mammalian, yeast, plant and insect cells). Several problems typically occur, however, when such cell types are used as host cells for heterologous gene expression. For example, recombinant polypeptides produced by expression of heterologous genes in eukaryotic cells are often rapidly degraded by proteases in the cells. Those polypeptides that do manage to accumulate when expressed in a cell can be disruptive to the normal machinery of the cell, thereby lowering the growth and/or survival rate and the polypeptide production ability of the cell.
Recombinant polypeptides produced by expression of heterologous genes in bacterial cells are usually found in the insoluble or xe2x80x9cinclusion bodyxe2x80x9d fraction of a bacterial lysate, rendering the bacterial cells and the polypeptides useless for biological and biochemical applications. Such inclusion bodies typically require further manipulation in order to solubilize and re-fold the recombinant polypeptide under conditions that must be determined empirically, resulting in further uncertainties. Moreover, recombinant polypeptides produced in bacterial cells often have low activity due to a failure of the polypeptides to assume a natural conformation and/or the inability of cells to glycosylate such polypeptides.
To overcome these problems, prior investigators have concentrated on improving the expression of recombinant polypeptides in bacterial cells using various xe2x80x9cfusion partnersxe2x80x9d linked to desired heterologous polypeptides to enable the expression and/or secretion of polypeptides in large amounts (U.S. Pat. No. 5,270,181, by McCoy et al., issued Dec. 14, 1993). Such fusion protein systems, however, fail to overcome the basic protein conformation problems encountered by expressing heterologous genes in a bacterial cell.
The use and purification of polypeptides produced by recombinant expression systems is a serious problem since recombinant production of polypeptides has become a mainstay in the development and production of therapeutic and diagnostic reagents. There is a continuing need for a simpler and commercially efficient method to produce substantial amounts of optimally active recombinant polypeptides for use in experimental, therapeutic and diagnostic applications.
General limitations in the commercial production of compounds (e.g., polypeptides) include problems relating to the efficiency of synthesis and, if a complex synthesis process is involved, the efficiency of recovery. The present invention provides a simpler and commercially efficient method to produce substantial amounts of a desired product molecule. The present invention provides a product and process for aggregating desired product molecules such that the desired molecules are sequestered in or within lipid bilayers. Such sequestration acts to protect the integrity of a product molecule, as well as to facilitate recovery of the molecule. In addition to the production and recovery of a desired product molecule, the novel sequestration method of the present invention can be used in a variety of applications. For example, a therapeutic composition can be produced by aggregating a desired product molecule in a plant cell, and the plant cell can be fed to an animal to administer the composition. Further uses of the present invention are described in detail below.
One aspect of the present invention includes a method to aggregate a desired product molecule in a lipid bilayer, comprising forming oligomers between two or more aggregate molecules that are physically associated with a lipid bilayer such that the aggregate molecules are accumulated while in association with the lipid bilayer. The accumulation of the aggregate molecules generates at least one membrane compartment having a morphology substantially similar to one or more membrane compartments shown in FIG. 4. Preferably the present method is performed in vivo and the steps of synthesis, aggregation and sequestration do not substantially interfere with cellular function.
Another aspect of the present invention includes a non-naturally occurring membrane housing compartment, comprising an intracellular structure inside of which proteins are sequestered without substantially interfering with cellular function. Preferred membrane housing compartments include stacked membrane structures and planar membrane structures. More preferred membrane housing compartments are formed by forming oligomers between a sufficient number of aggregate molecules such that a membrane compartment is formed that is substantially similar to the morphology of one or more membrane compartments shown in FIG. 4.
Yet another aspect of the present invention includes a nucleic acid molecule encoding an aggregate molecule comprising: a) an adhesive molecule capable of forming oligomers between two or more aggregate molecules, the adhesive molecule to a lipid bilayer; and b) a desired product molecule functionally associated with the adhesive molecule. A preferred nucleic acid molecule encodes at least a portion of xcex2-glucuronidase that is capable of forming an oligomer with another molecule. A more preferred nucleic acid molecule further comprises a nucleic acid sequence that encodes a transmembrane molecule comprising at least a portion of a coronavirus avian infectious bronchitis virus M protein. Also included in the present invention is a recombinant cell having a recombinant molecule comprising a nucleic acid molecule of the present invention operatively linked to an expression vector.
One embodiment of the present invention comprises a delivery vehicle comprising a recombinant cell having at least one nucleic acid molecule encoding a reagent and an aggregate molecule, the aggregate molecule being capable of physically associating with a lipid bilayer. The reagent can be chosen from antigens, toleragens, drugs (e.g., antibiotics or anti-neoplastic drugs), toxic compounds, markers, hormones, antibodies, cytokines and growth factors.
Another embodiment of the present invention includes a command secretion system, comprising: a) a recombinant cell comprising: (i) a nucleic acid molecule encoding an aggregate molecule comprising an adhesive molecule and a desired product molecule, in which the desired product molecule is linked to the adhesive molecule by a transmembrane molecule, the desired product molecule being covalently attached to a proteolytic restriction site; (ii) a nucleic acid molecule encoding a protease that is capable of cleaving the desired product molecule from the transmembrane molecule at the proteolytic restriction site; and b) an inducing agent capable of inducing the expression of the nucleic acid molecule encoding the protease.
Yet another embodiment of the present invention includes an assay for identifying protein associated molecules, comprising: a) forming a sequestered protein complex by contacting a first adhesive molecule attached to a first subunit of a protein formation, with a second adhesive molecule attached to a second subunit of the protein formation; b) contacting the sequestered protein complex with a putative associated molecule; and c) determining whether the putative associated molecule is capable of associating with the sequestered protein complex by assessing the binding of the putative associated molecule to the sequestered protein complex. Preferably, the first subunit is identical to the second subunit.
The present invention also includes a biological sensor system formed by the method comprising aggregating aggregate molecules that are physically associated with a lipid bilayer in such a manner that the aggregate molecules are accumulated in association with a portion of the lipid bilayer, the aggregate molecules being functionally associated with a biological sensing molecule. Preferably, the biological sensing molecule comprises a molecule capable of responding to a stimulus such as light or chemicals.
Another aspect of the present invention comprises a method to detoxify a cell, comprising: a) providing to a cell an adhesive molecule having at least one site capable of binding to a free-floating toxin; and b) contacting the adhesive molecule with the toxin, whereby said adhesive molecule and toxin form a complex that is less toxic to the cell than the free-floating toxin.
Yet another aspect of the present invention comprises a method for increasing the concentration of a desired product molecule within a cell, comprising: a) forming within a cell oligomers between two or more aggregate molecules having desired product molecules attached thereto, wherein the concentration of the desired product molecules in the cell exceeds the concentration of the desired product molecules in cells where the oligomers are not formed.
FIG. 1 schematically illustrates the interaction of aggregating molecules attached to a lipid bilayer on the cytoplasmic side of an organelle membrane.
FIG. 2 schematically illustrates the interaction of aggregating molecules attached to a lipid bilayer on the lumenal side of an organelle membrane.
FIG. 3 schematically illustrates the interaction of aggregating molecules not attached to a lipid bilayer.
FIG. 4A is an electron micrograph of a Zippered-membrane (Z-membrane) whorl formed in a tobacco plant cell.
FIG. 4B is an electron micrograph of forming Z-membrane whorls in which the zippering of adjacent ER membranes is seen.
FIG. 4C is an electron micrograph of Z-membranes formed in a tobacco cell and immunolabelled with antibodies specific for GUS.
FIG. 4D is an electron micrograph of a tobacco cell in which both stacked and whorled Z-membranes are seen.
FIG. 4E is an electron micrograph of Z-membranes formed in a yeast cell.
FIG. 4F is an electron micrograph of a yeast cell with stacked Z-membranes immunolabelled with antibodies specific for GUS.
FIG. 5 illustrates the results of sucrose density gradients of protoplasts correlated with various enzymatic functions.